Dendritic spines undergo synaptic pruning during adolescence, a process which is dysregulated in autism and schizophrenia. The mechanism underlying this synaptic pruning is not known, but is likely to involve reductions in NMDA receptor activation. We recently found that, during the pubertal period (~PND 35-45), extrasynaptic 4? GABAA receptors (GABARs) emerge on dendritic spines of CA1 hippocampal pyramidal cells of mice for ~10d. Moreover, activation of NMDA receptors is impaired in +/+ but not 4 or ?-/- mice, implicating 4? in the reduction of NMDAR function during puberty. In this proposal, we will test the hypothesis that synaptic pruning in adolescence is indeed due to this increased expression of 4? GABARs, and we will explore the cellular, circuit and behavioral consequences of this effect. The proposed experiments will use a wide combination of techniques, including two-photon imaging, two-photon uncaging, electrophysiological, pharmacological, anatomical and immunocytochemical assays to directly assess the functional outcome of this novel form of inhibition on the spines at puberty. Initially, optochemical techniques with RuBiGABA, a novel caged GABA compound, will be used to map GABAergic currents in spines at puberty. Preliminary data with this technique show that spines have GABAergic currents. Then, the impact of these receptors on NMDA receptor-induced Ca2+ influx from individual spines will be examined using two-photon Ca2+ imaging along with local uncaging of Rubi-GABA. These experiments will also use acute pharmacological (agonists, modulators) and genetic manipulation (knock-out, adenovirus-shRNA knock-down) of 4? to explore the role of these receptors in regulating NMDA receptor function. In parallel, to determine the impact of 4?-mediated inhibition during puberty on spine density post-pubertally, we will chronically manipulate 4? function (as above) over the 10 days when 4? receptors are elevated (~PND 35-45) and quantify spine density at 8-wks of age. Preliminary data shows that spine density is indeed decreased post-pubertally in +/+ but not 4-/- mice, implicating 4? GABARs in pubertal synaptic pruning. Both immunocytochemical and electrophysiological correlates of spine density changes will be examined. Finally, to explore the effect of this pruning on circuit and behavioral plasticity, we will also determine the impact of these alterations on synaptic plasticity (long-term potentiation (LTP), long-term depression (LTD), reversal of LTP), and on behaviorally flexibility in two hippocampal-dependent spatial learning tasks. The findings from the proposed studies will directly address the functional role of GABAergic inhibition on dendritic spines at puberty, and will also provide mechanisms for the process of synaptic pruning in adolescence, as well as explore functional outcomes of alterations in this process. These results will be relevant for cognitive disorders such as autism, where reduced synaptic pruning and reduced behavioral flexibility are associated with single nucleotide polymorphisms of the 4 gene, suggesting a genetic link.